Purification and characterization of canine urinary kallikrein

Abstract

The renal kallikrein-kinin system may play a role in the regulation of sodium and water balance. Although the dog is a frequently used experimental animal in the study of the renal kallikrein-kinin system, dog urinary kallikrein (DUKK) has been poorly studied. We have purified DUKK by a series of chromatographic and electrophoretic procedures including anion-exchange chromatography, filtration through p-aminobenz-amidine-Sepharose (to remove contaminating nonkallikrein esterases), gel filtration, isoelectric focusing, and molecular sieve HPLC. This DUKK preparation gave three protein bands on polyacrylamide gel electrophoresis, each having similar esterolytic and kininogenase activities and immunological identity. Preparative isoelectric focusing indicated the presence of multiple forms of kallikrein with p I's of 3.93, 4.05, 4.24, and 4.44, the species with a p I of 4.24 constituting the major component. Neuraminidase treatment converted all of the forms into the component with a p I of 4.44, suggesting the charge heterogeneity was due mainly to differences in sialic acid content. DUKK has a specific activity of 3 mg bradykinin eq/min/mg protein when partially purified dog kininogen is used as a substrate. It is a glycoprotein with a molecular weight of 40,500 (amino acid analysis best fit method) and an alkaline pH optimum (9.0–9.5). DUKK is resistant to soybean trypsin inhibitor and lima bean trypsin inhibitor but is inhibited by several serine protease inhibitors such as antipain, leupeptin, and p-aminobenzamidine. PhePheArgchloromethyl ketone is a very potent inhibitor of DUKK. Contrary to previous reports, DUKK is also inhibited by N-α- p-tosyl- l-lysine chloromethyl ketone and aprotinin, the inhibition by the latter being inversely related to the concentration of NaCl in the medium. The esterolytic and amidolytic activities of DUKK are inhibited by an increase in NaCl concentration of the medium. This inhibition may be related to a NaCl-induced conformational change in the enzyme moiety.